Efficient construction of rAAV-based gene targeting vectors by Golden Gate cloning. (May 2014)

• Record Type: Journal Article
• Authors: Luo, Yonglun; Lin, Lin; Bolund, Lars; Sørensen, Charlotte Brandt
• Is Part Of: Biotechniques. Volume 56:Number 5(2014)
• Page Start: 263
• Page End: 268
• Publication Date: 2014-05
• Subjects: gene targeting -- rAAV -- Golden Gate cloning -- p53 -- tau; Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/000114169
• Languages: English
• ISSNs: 0736-6205

Abstract

The recombinant adeno-associated virus (rAAV) has proven to be an efficient and attractive tool for targeted genome engineering. Here we present a novel method employing the Golden Gate cloning strategy for fast and efficient construction of rAAV-based gene knockout or single-nucleotide knockin vectors. Two vectors, pGolden-Neo and pGolden-Hyg, were generated as common assembling modules to confer antibiotic resistance to the targeting vector. To validate the method, we then generated two rAAV-based targeting vectors: pAAV-pTP53-KO and pAAV-hTauP301L-KI. Furthermore, we generated a pGolden-AAV plasmid that allows one-step generation of an rAAV-based targeting vector. Our new methodology for rAAV targeting vector assembly is efficient, accurate, time-saving, and cost-effective.